Copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) has gained widespread use in chemical biology for applications such as labeling of biomolecules in complex mixtures and imaging of fixed cells and tissues. (Kolb, et al., Angew. Chem. Int. Ed. 2001, 40, 2004; Rostovtsev, et al., Angew. Chem. Int. Ed. 2002, 41, 2596; Wu and Fokin, Aldrichimica Acta 2007, 40, 7.) Incorporation of fluorescent probes into proteins, DNA, RNA, lipids and glycans within their native cellular environments provides opportunities for imaging and understanding their roles in vivo. (Best, Biochemistry 2009, 48, 6571.)
For example, glycans in proteins are displayed on the cell surface with implications in numerous physiological and pathological processes. Aberrant glycosylation on the surface of diseased cells is often observed in pathological conditions, such as inflammation and cancer metastasis. In particular, altered terminal sialylation and fucosylation, which are believed to result from changes in expression locations and levels of sialyltransferases and fucosyltransferases, are associated with tumor malignancy. The ability to explore the biological information content of glycans as biomarkers of cancer, attached to either proteins or lipids, has become a major course of glycomics research. (Hsu, et al., Proc. Nat. Acad. Sci. U.S.A., 2007, 104, 2614; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371.)
Analysis of changes in glycosylation patterns in living systems is now possible. (Prescher and Bertozzi, Nat. Chem. Bio. 2005, 1, 13.) Metabolic incorporation of an unnatural carbohydrate containing unique functional group that acts as a bioorthogonal chemical reporter into the cell biosynthetic machinery initiates the process. The modified glycan is then processed and constructed on the cell surface. Subsequent reaction with a detectable fluorescent probe equipped with a complementary bioorthogonal functional group enables detection of the incorporated unnatural glycan (FIG. 1). (Sletten and Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974-98.)
The concept of bioorthogonal chemical reporter has been applied to proteomic analysis of glycosylation in proteins and chemical remodeling of cell surfaces in living systems. Bioorthogonal chemical reactions have also been used for other applications, such as protein labeling, activity-based protein folding, protein targets identification, posttranslational modifications, and cell proliferation monitoring. Labeling of specific functional groups on living cell via bioorthogonal chemical reporter strategies have become increasingly powerful in cell biology. These approaches are often based on cycloadditions as ideal bioorthogonal reactions because of their intrinsic selectivity and tunable electronics. However, there are still many challenges facing the field. For example, most bioorthogonal reporter strategies entail multistep procedures that use fluorophroe-labeled reactant partners, which often cause high background fluorescent noise that is difficult to remove from intracellular environments or tissues. In addition, these methods require high concentrations of reagents and catalysts in order to achieve detectable signals.
Some recent efforts have been focused on the design of fluorogenic CuAAC reactions between non-fluorescent alkyne and azide, which can ligate to afford a highly fluorescent triazole complex (FIG. 1). (Sivakumar, et al., Org. Lett. 2004, 24, 4603; Sawa, et al., Proc. Nat. Acad. Sci. U.S.A., 2006, 103, 12371; Xie, et al., Tetrahedron 2008, 64, 2906; Li, et al., Org. Lett. 2009, 11, 3008; Le Droumaguet, et al., Chem. Soc. Rev. 2010, 39, 1223; Chao, et al., Sci. China Chemistry 2012, 55, 125.) This type of CuAAC reaction occurring in high efficiency would have broad applications in the emerging field of cell biology and functional proteomics due to the distinct fluorescence properties in formation of the triazole without background fluorescent noise of the starting materials. Unfortunately, the use of CuAAC reactions in living systems has been hindered because the reactions require toxic copper(I) ion as the catalyst.
To circumvent the cytotoxicity associated with metal catalyst, the ring strain-promoted azide-alkyne cycloadditions (SPAAC) without using metal catalyst have been developed. (Agard, et al., J. Am. Chem. Soc. 2004, 126, 15046; Codelli, et al., J. Am. Chem. Soc. 2008, 130, 11486; Debets, et al., Acc. Chem. Res. 2011, 44, 805; Dommerholt, et al., Angew. Chem. Int. Ed. 2010, 49, 9422; Friscourt, et al., J. Am. Chem. Soc. 2012, 134, 5381; Jewett, et al., J. Am. Chem. Soc. 2010, 132, 3688; Ning, et al., Angew. Chem. Int. Ed. 2008, 47, 2253; Poloukhtine, et al., J. Am. Chem. Soc. 2009, 131, 15769; Varga, et al., Chem. Eur. J. 2012, 18, 822.) A cyclooctyne moiety is often incorporated as a stem structure into the SPAAC reagents, such as difluorinated cyclooctynes (DIFO) and the derivatives. An attempt toward this approach was reported by Bertozzi and co-workers using the biarylazacyclooctynone ring fused with a coumarin fluorophore (J. C. Jewett, C. R. Bertozzi, Org. Lett. 2011, 13, 5937-5939). Although the compound undergoes a cycloaddition reaction with 2-azidoethanol to give a 10-fold increase in fluorescence intensity, the triazole product exhibited a low quantum yield (Φf=0.04) and required relatively high energy excitation (˜300 nm), making it unsuitable for imaging in living systems.